ARTICLE Loss of Function in Phenylketonuria Is Caused by Impaired Molecular Motions and Conformational Instability Søren W. Gersting, 1 Kristina F. Kemter, 1 Michael Staudigl, 1 Dunja D. Messing, 1 Marta K. Danecka, 1 Florian B. Lagler, 2 Christian P. Sommerhoff, 3 Adelbert A. Roscher, 1 and Ania C. Muntau 1, * A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiop- terin (BH 4 ), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH 4 treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH 4 -responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was dis- turbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding—with particular impact on the regula- tory domain—observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a pro- tein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein- folding disease with loss of function. Introduction Deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1) causes phenylketonuria (PKU [MIM 261600]) and is the most common inborn error of amino acid metab- olism in European-descended populations. Since the intro- duction of a dietary treatment fifty years ago, PKU has been the prototype for a treatable genetic disease and, later, for genetic screening in human populations. 1 The recent recog- nition of a new pharmacologically treatable phenotype of PAH deficiency challenged the classical view of hereditary diseases that result in loss of enzyme function. We previ- ously showed that a significant share of PKU patients re- sponds to oral administration of the natural PAH cofactor (tetrahydrobiopterin, BH 4 ) although these individuals do not display biochemical evidence of BH 4 deficiency. The treatment reduces blood phenylalanine concentrations, restores enzyme activity in vivo, and significantly increases dietary-protein tolerance. 2 Some authors proposed that restoration of enzyme function observed under treatment with pharmacological doses of BH 4 is transmitted by correc- tion of PAH misfolding. 2–5 About 80% of all mutations in the PAH gene are missense, and experimental data on the conformational impact of single amino acid replacements on allostery, sta- bility, and folding of the PAH protein is scarce. Because the crystal structure of full-length PAH has not yet been com- pletely solved, 6–8 a composite model is commonly used for 3D structural analysis. 9 PAH is a homotetrameric enzyme, with each subunit composed of three functional domains: the N-terminal regulatory domain (residues 1–142); the catalytic domain (residues 143–410), which includes binding sites for substrate and cofactor; and the oligomerization domain at the C terminus (residues 411– 452). There is a high degree of structural interplay between the single domains and the subunits, respectively. 6–8,10–13 Substrate (L-phenylalanine) and cofactor binding induce conformational changes, which are transmitted through networks of side-chain interactions. 14,15 This is the basis for homotropic allostery that allows for fine-tuned regula- tion of PAH enzyme activity comprising substrate activa- tion, modulation of oligomerization, and the affinity to substrate and cofactor. 3,4,12,15 We hypothesized that the structural flexibility of PAH permits gross conformational changes required for enzyme function and thus renders the enzyme susceptible to protein misfolding. This view was supported by previous studies demonstrating dis- turbed oligomerization and accelerated degradation of some variant PAH proteins. 3,4,16–19 However, only little is known about the structural mechanisms leading to loss of function in phenylketonuria and in other inherited diseases that exhibit loss-of-function pathogenesis. The aim of this study, therefore, was to elucidate the molecular basis of loss of function in PKU. The impact of single side-chain replacements associated with BH 4 -re- sponsiveness on function and conformational stability of 1 Department of Molecular Pediatrics, Children’s Research Center, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University, 80337 Munich, Germany; 2 Department of Medical Genetics, Molecular and Clinical Pharmacology, Innsbruck Medical University, 6020 Innsbruck, Austria; 3 Department of Clinical Chemistry and Clinical Biochemistry, Surgical Clinic, Ludwig Maximilians University, 80337 Munich, Germany *Correspondence: ania.muntau@med.uni-muenchen.de DOI 10.1016/j.ajhg.2008.05.013. ª2008 by The American Society of Human Genetics. All rights reserved. The American Journal of Human Genetics 83, 5–17, July 2008 5